Live, avirulent Salmonella choleraesuis vaccine used for inducing an immune response in pigs

ABSTRACT

Disclosed herein are methods for attenuating virulent gram negative bacteria to produce avirulent bacteria. The methods comprise passaging the wild-type bacteria through phagocytic cells, such as macrophages or polymorphonuclear leukocytes, or through lysosomes derived from such cells, a sufficient number of times until the bacteria become avirulent to the animal host. The bacteria are preferably from the family Enterobacteracea and most preferably from the genus Salmonellae. The invention further comprises the avirulent bacteria produced by the methods, pure cultures of such bacteria, and methods of using the bacteria, preferably in a vaccine for administration to an animal host to induce an immune response to the wild-type gram negative bacteria in the host.

This application is a continuation of Ser. No. 07/773,429 filed Oct. 9,1991, now abandoned, which is a continuation-in-part of application Ser.No. 07/607,662 filed Nov. 1, 1990, now abandoned.

FIELD OF THE INVENTION

This invention relates generally to a bacterial attenuation method andto vaccine production. Specifically, it relates to a method ofattenuating gram negative bacteria to produce avirulent bacteria thatare useful in vaccines for humans and animals. One such strain has beenfield tested and found to have effectively prevented salmonellosis inswine and pigs with no reversion to virulence.

BACKGROUND OF THE INVENTION

Gram negative bacteria cause a wide variety of diseases in humans andanimals. These include plague, caused by Yersinia pestis, typhoid fever,caused by Salmonella typhi, gonorrhea, caused by Neisseria qonorrhoeae,dysentery, caused by Shigella dysenteriae, gastroenteritis, commonlycaused by Salmonella typhimurium, Escherichia coli, and Campylobacterjejuni, bacterial sepsis, caused primarily by Escherichia coli,pseudomonas aeruginosa, and Klebsiella pneumoniae, and septicemicdiseases in cattle and pigs, caused by Salmonella dublin and Salmonellacholeraesuis, respectively.

Humans and animals have evolved many defenses to infection by gramnegative bacteria. One of the first lines of defense are the body'sphagocytic cells. These cells engulf invading microorganisms and killthem by various methods, such as the release of proteolytic enzymes andoxygen radicals.

Unfortunately, many types of bacteria have evolved means to inhibit orresist the many microbicidal substances in phagocytic cells, therebyallowing them to survive within the cells. Such facultativeintracellular pathogens are a clinically important group of bacteria.They include bacteria from the genera Salmonelia, Yersinia, Shigella,and Neisseria.

Because the intraphagocytic environment is so hostile to bacteria, itseems reasonable to assume that the selection of bacteria from withinphagocytes would follow the Darwinian principle of survival of thefittest. Current reports indicate that, in order to survive inphagocytes, Salmonellae must possess virulence attributes, such asplasmids, porins, and other outer membrane proteins related tovirulence. See Taira, et al., Microbial Pathogenesis, 7:165-173 (1989),Tufano, et al., Microbial Pathogenesis, 7:337-346 (1989), and Gulig,Microbial Pathogenesis, 8:3-11 (1990), all of which are incorporatedherein by reference. It has also been reported that mutants unable tosurvive in macrophages have lost immunogenicity and virulence whencompared to their parental strains. See Buchmeier, et al., Infection andImmunity, 57:1-7 (1989), Fields, et al., Science, 243:1059-1062 (1989),and Buchmeier, et al., Science, 248:730-732 (1990), all of which areincorporated herein by reference. Therefore, the reasonable expectationwould be that Salmonellae able to survive in phagocytic cells wouldpossess optimal virulence properties.

Surprisingly, the inventor has discovered the opposite result to thatexpected. Salmonellae that survived serial passages through livephagocytic cells or their lysosomal products exhibited decreasedvirulence and, after a sufficient number of passages, became avirulent.The avirulent bacteria still produced an immunogenic response wheninnoculated into an animal host, thereby providing the basis forvaccines against gram negative bacteria.

Such vaccines would be highly desirable because such bacteria,particularly the faculatative intracellular pathogens, are often able toevade the body's defense mechanisms. A vaccine would prepare and enhancethe defense mechanisms prior to significant invasion by the bacteriaagainst which the vaccine is directed. Live, avirulent bacteria, asopposed to killed bacteria or inactivated toxins, are particularlydesirable as vaccines because they usually provide a broader immunesystem response.

SUMMARY OF THE INVENTION

It is an object of the invention to provide methods of attenuating gramnegative bacteria that are virulent to an animal host, thereby producingavirulent gram negative bacteria.

A further object of the invention is to provide avirulent, gram negativebacteria.

A still further object of the invention is to provide a method andvaccine for inducing an immune response in an animal host to gramnegative bacteria.

Another object of the invention is to provide a method and vaccine forprotecting an animal host against gram negative bacteria.

Additional objects and advantages of the invention will be set forth inpart in the description that follows, and in part will be obvious fromthe description, or may be learned by the practice of the invention. Theobjects and advantages of the invention will be attained by means of theinstrumentalities and combinations particularly pointed out in theappended claims.

To achieve the objects and in accordance with the purpose of theinvention, as embodied and broadly described herein, the presentinvention provides methods of attenuating gram negative bacteria thatare virulent to an animal host. In the preferred embodiment, thevirulent, wild-type gram negative bacteria are passaged throughphagocytic cells a sufficient number of times until the bacteria becomeavirulent to the host but are still immunogenic. Preferably, thephagocytic cells are polymorphonuclear leukocytes (PMNLs). In analternative embodiment, the wild-type gram negative bacteria arepassaged through cultures of lysosomes obtained from phagocytic cells asufficient number of times until the bacteria become avirulent to thehost but are still immunogenic. Preferably, the lysosomes are obtainedfrom PMNLs.

The invention further comprises avirulent gram negative bacteriaproduced by the methods of attenuation described herein. Preferably, thevirulent gram negative bacteria are selected from the familyEnterobacteracea. Most preferably, the bacteria are selected from thegenus Salmonella.

The invention further comprises an avirulent strain of Salmonellacholeraesius. The strain metabolizes both glycerol and d-xylose,exhibits increased resistance to being killed by neutrophils andhydrogen peroxide as compared to wild-type Salmonella choleraesuisstrains, and is noninvasive to Vero cells.

The invention further comprises a vaccine and method for inducing animmune response to gram negative bacteria in an animal host. A vaccinecomprising an immunologically effective amount of the avirulent bacteriaof the invention in a pharmaceutically acceptable carrier isadministered to the animal host.

Other features of the invention will be apparent from the DetailedDescription and the figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of the daily rectal temperatures of 5pigs given neutrophil adapted S. choleraesuis strain 54 and 5 pigs giventhe virulent parent strain 38 per os.

FIG. 2 is a graphic representation of the daily rectal temperatures of 9pigs vaccinated with live S. choleraesuis strain 54 per os andchallenged with a virulent field strain of S. choleraesuis and of 6challenge control pigs. All pigs were challenged with 10⁹ CFUs per os.

FIG. 3 is a graphic representation of the daily weight changes of 9 pigsvaccinated with S. choleraesuis strain Scs 54 and of 6 challenge controlpigs after challenge with a virulent field strain. Weight changesbetween vaccinated and challenge control pigs were significantlydifferent one week as well as two weeks after challenge (P<0.01) by x².

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the presently preferredembodiments of the invention, which, together with the followingexamples, serve to explain the principles of the invention.

The invention relates to methods for attenuating virulent, wild-typegram negative bacteria, to the resulting attenuated or avirulentbacteria, and to vaccines containing such bacteria. As used herein, theterm "virulent" pertains to bacteria that are capable of invading thetissues of an animal host and causing disease or other pathologiceffects. Virulence is measured by severity of disease and can bequantitated by the median lethal dose (LD₅₀) in experimental animals,the numbers of organs (generally spleen or liver) colonized by thebacteria, and the colony forming units (CFUs) from the infected organs.As used herein, the term "attenuated" refers to the weakening ordecreasing of the virulence of the wild-type bacteria. As used herein,the term "avirulent" refers to previously virulent bacteria that havebeen attenuated to a sufficient degree that they are no longer virulentto their natural animal host. Thus, their administration to the animalhost would not cause disease. Generally, such avirulence can be shown bya decrease in the LD₅₀, the numbers of colonized organs, or the numberof CFUs by a factor of 10, preferably by a factor of 100, and mostpreferably by a factor of 1000.

The methods of the invention are directed to attenuating gram negativebacteria that are virulent to an animal host to a sufficient degree toproduce gram negative bacteria that are avirulent to the host. In thepreferred embodiment, the method comprises serially passaging thevirulent, wild-type gram negative bacteria through phagocytic cells asufficient number of times until the bacteria are rendered avirulent tothe animal host while still being immunogenic. In particular, thebacteria are mixed and incubated with the phagocytic cells, underconditions conducive to the maintenance and growth of the bacteria, fora sufficient period of time for most of the bacteria to be phagocytizedby the cells. The amount of bacteria and cells and the time,temperature, and other conditions for passaging will be known to thoseskilled in the art or easily determinable without undue experimentation,given the teachings contained herein. Preferably, the bacteria are mixedwith the cells in a ratio from about 100 to 1 to about 5 to 1 at atemperature of about 35° C. to 40°0 C. for approximately 30 to 60minutes. Most preferably, the ratio of bacteria to cells isapproximately 10:1, the temperature is about 37° C., and the time ofincubation is approximately 45 minutes. Phagocytosis of the bacteria isfacilitated by the addition of specific antibody in a sub-agglutinatingconcentration (usually diluted 1:100 to 1:200). The reactants arepreferably suspended in a tissue culture medium (M199) containing 10%fetal calf serum.

The unphagocyted bacteria are then separated from the phagocytic cells.Preferably, this is done by killing the unphagocytized bacteria bycontacting the mixture with an antibiotic agent that does not harm thephagocytic cells. Preferred antibiotics include gentamicin andkanamycin. The amount or concentration of antibiotic and the time it iscontacted with the mixture will be known to those skilled in the art oreasily determined without undue experimentation, given the teachingscontained herein. Preferably, the concentration of antibiotic is fromabout 25 to about 150 ug (micrograms), and the contact time is fromabout 15 to about 45 minutes. Most preferably, the concentration isabout 100 ug, and the contact time is about 30 minutes.

The phagocytized bacteria are recovered from the phagocytic cells bydisrupting the cells and separating the bacteria from the cellulardebris. The phagocytic cells can be disrupted mechanically, by osmoticlysis, or chemically. Preferably, disruption is by exposure to a 0.2%solution of saponin for about 10 to 30 seconds. The period of time fromthe beginning of the passaging step to the beginning of the recoverystep is approximately from 60 to 120 minutes.

The number of serial passages necessary for obtaining avirulent bacteriawill vary somewhat, depending upon the nature of the starting bacteria,the nature of the phagocytic cells, and the type of animal host, but thenumber will be readily determinable without undue experimentation bypersons skilled in the art, given the teachings contained herein.Generally, the number of passages will be at least 5, preferably 8, andmost preferably 12.

As mentioned above, the bacteria are passaged through phagocytic cells.Such cells include macrophages and microphages. Of the microphages,PMNLs (also called neutrophils) are the ones that engulf bacteria.Therefore, they are the only microphages that should be deemed to comewithin the scope of the term phagocytic cell as used herein. Moreover,PMNLs are the preferred cells for use in this method of the invention.

It is preferred that the phagocytic cells are free of any bacteriabefore passaging is initiated, and it is highly preferred that the cellsbe free of the same type of bacterium as that bacterium which is to bepassaged through the cells. Such freedom from contamination can bedetermined by techniques known in the art.

In an alternative embodiment of the invention, the method forattenuating the bacteria comprises passage through lysosomes instead ofphagocytic cells. The lysosomes are organelles of the phagocytic cellsthat contain most, if not all, of the many bactericidal compounds thatsuch cells use to destroy foreign microorganisms.

The lysosomes are obtained from phagocytic cells by known techniques.Preferably, the phagocytic cells are PMNLs. The wild-type, virulent gramnegative bacteria are then passaged through mixtures of such lysosomesfor a sufficient number of times until the bacteria become avirulent tothe animal host while still being immunogenic. The passaging comprisesmixing and incubating the bacteria with the lysosomes, under conditionsconducive to the growth of the bacteria, and then separating thebacteria from them using known techniques, such as centrifugation.Repeated serial passaging results in the eventual recovery of avirulentbacteria. Fresh lysosomes are used for each passage. Preferably, thebacteria are grown by culturing in a bacterial growth medium after eachrecovery from the lysosomes and prior to the next passage through thefresh lysosomes.

The relative amounts of bacteria and lysosomes, the times the bacteriaare in contact with the lysosomes and the culture medium, and thetemperature and other conditions for passaging will depend primarilyupon the particular type of gram negative bacteria and the particularanimal host. However, these and other conditions will be known to thoseskilled in the art or easily determinable without undue experimentation,given the teachings contained herein. Preferably, the bacteria are mixedwith the lysosomes in a volume ratio from about 1 to 5 to about 1 to 20at a temperature of about 37° C. to about 42° C. for approximately 30 to60 minutes. Most preferably, the ratio of bacteria to lysosomes isapproximately 1 to 10, the temperature is about 37° C., and the mixingtime is approximately 30 to 45 minutes.

The number of serial passages necessary for obtaining avirulent bacteriawill vary somewhat, depending upon the nature of the starting bacteria,the nature of the phagocytic cell from which the lysosomes are obtained,and the type of animal host, but the number will be readily determinablewithout undue experimentation by persons skilled in the art, given theteachings contained herein. Generally, the number of passages will befrom about 5-15, preferably about 13.

As mentioned above, the method of the invention may be applied to anygram negative bacteria. Such bacteria have similar virulencecharacteristics and invasion strategies. They also have very similarouter membrane structures, including pill and adhesion proteins. Thepreferred animal hosts are any animals that may be infected by suchbacteria. These include, but are not limited to, humans and otherprimates or mammals, cattle, swine, birds, and fish.

Within the category of gram negative bacteria, a preferred subcategoryare bacteria from the family Enterobacteriacea. Many types of bacteriafrom this family infect humans and many animals. Particularly importantwithin this family are Salmonellae Shigellae, Klebsiellae,Eschericheriae, and Yersiniae. These bacteria are particularlytroublesome to humans and commercially important animals, such as cattleand swine. Particularly important species within these genera include S.typhi, S. choleraesuis, S. dublin, S. enteritidis, S. typhimurium,Shigella. dysenteriae, K. pneumoniae, E. coli, and Y. pestis.

Another important subcategory of gram negative bacteria are thefacultative intracellular pathogens. These include the genera Neisseriaand Brucellae and the previously mentioned genera Salmonella, Yersinia,and Shiqella. Within the first two genera, particularly importantspecies include N. gonorrhoeae and B. abortus.

Still other important gram negative bacteria are those from the generaPseudomonas and Haemophius particularly the species P. aeruginoa, H.spp, and H. influenzae.

The attenuated or avirulent gram negative bacteria produced by themethods of the invention are encompassed within the invention.Preferably, such bacteria are in pure culture, and, accordingly, theinvention encompasses compositions of matter comprising such purecultures. As used herein, the term "pure culture" means a compositioncomprising the bacteria in a culture medium, wherein the mixture is freeof other microorganisms.

For both methods of the invention, the preferred bacteria are from thegenus Salmonellae. Within this genus, the preferred species are S.choleraesuis and S. dublin, which infect pigs and cattle, respectively,and S. enteritidis, which infects many host species, including humansand birds. The most preferred starting bacterial variety is S.choleraesuis var. Kunzendorf strain 38, which gives rise to theavirulent strain S. choleraesuis var. Kunzendorf strain 38 PMNa. Strain38 is preferably used because of its virulence and ability to fermentglycerol (+) which can then be used as a marker for recovery. Both theparent and vaccine strains ferment glycerol which thus distinguishesthem from other S. choleraesuis strains.

S. choleraesuis var. Kunzendorf strain 38 PMNa is distinguished from itswild-type parent by the absence of the virulence plasmid exhibited bythe parent and by being able to grow on a medium containing d-xylose,which it metabolizes. Additionally, it exhibits an overall increasedresistance to PMNL killing and to killing by hydrogen peroxide, and itwas non-invasive in a Vero cell assay. A pure culture of this avirulentstrain was deposited under the Budapest Treaty on Oct. 29, 1990 in thepermanent collection of the American Type Culture Collection (ATCC),12301 Parklawn Drive, Rockville, Md. U.S.A. 20852 and assigned accessionnumber 55105.

Given the teachings contained herein and this particular strain, personsskilled in the art can use known techniques to obtain mutants andderivatives that still have the utility as immunogens in antimicrobialvaccines as does the parent avirulent strain (Kunzendorf strain 38PMNa). For example, such mutants or derivatives may have differentnutritional requirements, different resistance to neutrophil killing andkilling by hydrogen peroxide or may exhibit different degrees ofnon-invasiveness in Vero cell assays. However, as long as they arederived from the strain and have immunogenic activity, they are withinthe scope of this invention.

The avirulent bacteria of the invention are expected to have utility asimmunogens in antimicrobial vaccines for animals, including birds, fish,cattle, swine, horses, mammals and primates in general, and humans. Suchvaccines can be prepared by techniques known to those skilled in theart, given the teachings contained herein. Such a vaccine would comprisean immunologically effective amount of the avirulent bacteria of theinvention in a pharmaceutically acceptable carrier. The vaccine could beadministered in one or more doses. An immunologically effective amountis determinable by means known in the art without undue experimentation,given the teachings contained herein. The amount of avirulent bacteriashould be sufficient to stimulate an immune response indisease-susceptible animals while still being avirulent. This willdepend upon the particular animal, bacteria, and disease involved. Therecommended dose to be administered to the susceptible animal ispreferably about 10⁷ -10⁹ bacteria/Kg of body weight and most preferablyabout 10⁸ bacteria/Kg of body weight. The carriers are known to thoseskilled in the art and include stabilizers and diluents. Such a vaccinemay also contain an appropriate adjuvant. The vaccine preparations mayalso be desiccated, for example, by freeze drying for storage purposesor for subsequent formulation into liquid vaccines.

Accordingly, the invention also comprises a method for inducing animmune response to virulent, wild-type gram negative bacteria in ananimal host for the purpose of protecting the host from such bacteria.The method comprises administering an immunologically effective amountof the avirulent gram negative bacteria of the invention to the hostand, preferably, administering the vaccine of the invention to the host.

The vaccines may be administered to animals by various routes, includingoral, intramuscular, subcutaneous, and intranasal. The preferred routeof administration is oral.

In the preferred embodiment of the invention, the vaccine comprisesavirulent S. choleraesis produced by the method of the invention. Itwould contain about 10⁸ bacteria in sterile water per kilogram of bodyweight. It would be administered orally in a duodenal capsule.

The avirulent bacteria produced by the method of the invention are alsouseful as reagents for scientific research on the properties ofpathogenicity, virulence, and infectivity of gram negative bacteria, aswell as host defense mechanisms. A composition in accordance with thepresent invention useful as an investigational reagent contains anamount of avirulent bacteria effective to provide the information oranalysis sought. The determination of the amount necessary to accomplisha particular research goal depends upon the specific type ofinvestigation involved and is readily within the routine skill of oneengaged in such research.

It is to be understood that the application of the teachings of thepresent invention to a specific problem or environment will be withinthe capabilities of one having ordinary skill in the art in light of theteachings contained herein. Examples of the products of the presentinvention and processes for their use appear in the following examples.

EXAMPLE 1 Selection for Avirulence of Salmonellae in PolymorphonuclearLeukocytes

The fate of bacteria that survive in polymorphonuclear leukocytes(PMNL)s and the changes that occur in the surviving bacterial populationhave not been extensively documented. In order to detect the effect ofPMNL residency on virulence properties of Salmonellae, Salmonellae wereserially recovered from PMNLs. Compared to the source strains,PMNL-adapted S. choleraesuis, S. dublin, and S. enteritidis invadedtissue culture cells, such as VERO cells, about 10 times lesseffectively than the source strain, and became totally or almost totallyavirulent for mice and pigs. Virulence was measured by LD₅₀, numbers ofspleens colonized with Salmonellae, and colony forming units (CFU) frominfected spleens. Salmonellae adapted to PML were immunogenic.

Materials and Methods

Selection of S. choleraesuis (SC.) and other Salmonellae 38 from porcinePMNLs. Scs 38 was grown overnight on trypticase soy agar. A singlecolony was suspended in phosphate buffered saline solution, and thesolution adjusted to a concentration of 2×10⁸ CFUs. Porcine PMNLs wereisolated from venous blood by lysis of erythrocytes and centrifugationin a Ficol-Hypaque gradient density to separate the PMNLs fromlymphocytes. The PMNL suspension was adjusted to a 5×10⁷ cells/ml inphosphate buffered saline and resuspended in medium M199 containing 10%fetal calf serum. Equal volumes of Scs 38 and PMNLs were incubated for45 min at 37° C. After centrifugation at 1600 rpm, the PMNL pellet wasresuspended in PBS containing 100 ug gentamicin and 100 ug kanamycin andincubated for 30 min. Following centrifugation, the PMNL supernatant wasinoculated on MacConkey agar and incubated for 48 hours. The pellet wassuspended in a 0.1% solution of SDS (or preferably in 0.2% saponin) inPBS to disrupt PMNLs, and immediately cultured on MacConkey agar. It wasimportant to insure that all Scs 38 recovered after this procedureoriginated from surviving phagocytosed bacteria, and did not include Scs38 that somehow survived on the surface of PMNLs or in the antibioticmedium. Only treatments with no growth in the supernatant fraction after48 hours incubation were therefore considered successful passages of Scsin PMNL. Certain strains were subjected to multiple PMNL exposure. Thus,following culture on MacConkey agar, after exposure to PMNLs asdescribed above, individual colonies were selected and resuspended to2×10⁸ colony forming units (cfu)/ml. The above-described PMNL exposureprocedure was then repeated for varying number of times, i.e., five andseven. Following multiple passage completion, a single glycerol (+)clone was selected and designated.

Selection of S. choleraesuis (Scs) and other Salmonellae 38 from porcineLysosomes. Lysosomes were extracted from PMNL by freeze-fracturing PMNLsafter their purification and centrifuging the debris at 1,600 g for 20min. The supernatant was concentrated through an Amicon membrane andsterile filtered through a Millipore membrane filter. Equal volumes ofSalmonellae were alternatively incubated for periods of 30 min in thelysosome extract and trypticase soy broth (30 min in lysosome extractfollowed by 30 min in trypticase soy broth etc). Only the lysosomeincubation was counted as a cycle. That is, a 13× extraction consistedof 13 lysosomal incubation and 13 trypticase soy broth incubationperiods.

Mouse inoculations. Swiss Webster mice were inoculated with specifiednumbers of Salmonellae into the left footpad by subcutaneous injection,using a tuberculin syringe fitted with a 26" needle. The injectionvolume was 80 ul. The mice were sacrificed, and the spleens wereharvested, homogenized, and cultured for Salmonellae using amicrodilution technique. Data were pooled from several experiments.Lethal dose 50's (LD₅₀) were computed according to the formula of Reedand Muench.

Pig inoculations. Pigs were infected with Scs 38 and its PMN-adaptedderivative by oral gavage, after 24-hour fasting. The inoculums weresuspended in phosphate buffered saline to neutralize stomach acid.

Results and Discussion

Salmonella choleraesuis var Kunzendorf strain 38 (Scs 38) was chosen forthe experiments because of its strong and well-defined virulenceproperties. See Griffith, et al., Am. J. Vet. Res., 45:1342-1348 (1984)and Finlay, et al., J. Cell. Biol., 107:221-230 (1988), both of whichare incorporated herein by reference. The virulence of Salmonellaeserially exposed to live porcine PMNLs or their lysosomal products wasexamined. Virulence for mice decreased with subsequent exposures ofSalmonellae to PMNLs or lysosomes (Table 1). Virulence, judged by LD₅₀and ability to invade and grow in the mouse spleen, was graduallydecreased and ultimately abolished when mice were infected with S.choleraesuis subjected to serial passages through pig PMNLs (Table 1).Similarly, virulence was abolished when S. choleraesuis was exposedserially 13× to PMNL lysosomal extracts (Table 1). When S. choleraesuiswas fed by gavage to pigs, the natural hosts of S. choleraesuis, thePMNL-adapted Salmonellae caused no clinical ill effects, whereas thewild source type caused high fever, anorexia, and diarrhea (data notshown). Death and multiple organ infections with high numbers ofSalmonellae occurred in the group of pigs that received the wild type,whereas no death occurred, and organ infection was minimal, in pigsreceiving PMNL-adapted Salmonellae (Table 3).

The investigation was then extended to S. dublin and S. enteritidis. S.dublin is a cattle-adapted pathogen, causing severe enteritis andsepticemia, primarily in dairy calves. Salmonella enteritidis is notadopted to a particular host. It occurs commonly in eggs and poultry andis the cause of most current human Salmonella food poisonings in theU.S.A. The LD₅₀ of the PMNL-adapted S. choleraesuis, S. dublin, and S.enteritidis was at least 3 logs higher than the LD₅₀ of the respectivesource strains (Table 2). The number of spleens infected, and the numberof Salmonellae per infected spleen, was considerably lower in groups ofmice that were infected with the PMNL-adapted respective Salmonellae(Table 4). In addition to loss of virulence, PMNL selection ofSalmonellae resulted in strong protective immunity (Table 5). Immunitywas dose-dependent, and was accompanied by the persistence in lownumbers of the immunizing PMNL-adapted S. choleraesuis (Table 5).

Salmonella choleraesuis and S. dublin cause in their host species asepticemic disease not unlike human typhoid fever. The role of PMNLs istherefore particularly important in host defenses to these diseases, toprevent the systemic spread of salmonella infection. The aboveobservations raise the possibility that PMNLs are "the first line ofdefense" not only because of their microbicidal role, but also becausethey select less virulent lineages from a heterogeneous infectingpopulation of bacteria. The mechanism of the attenuated virulence is notknown.

                                      TABLE 1                                     __________________________________________________________________________    Selection of S. choleraesuis (Scs) 38 from porcine PMNL. Data                 were pooled from 3 experiments.                                                                  Killed   No spleens                                                                          Log.sub.10 Scs/                             Inocula:                                                                           Dose     No Mice                                                                            days pi                                                                           No Dead                                                                            infected                                                                            spleen                                      __________________________________________________________________________    Virulent source strain: S. choleraesuis 38                                    Scs 38                                                                             5.0 × 10.sup.1                                                                   8     8  0    5/8   7.74 ± 1.12                              Scs 38                                                                             1.6-5.0 × 10.sup.1                                                               19   14  3    18/19 6.17 ± 0.65                              Scs 38                                                                             1.6-2.6 × 10.sup.2                                                               26    8  6    23/26 6.10 ± 0.92                              Scs 38                                                                             1.6 × 10.sup.2                                                                   9    14  1    9/9   4.02 ± 0.16                              Scs 38                                                                             1.6 × 10.sup.3                                                                   10    8  4    10/10 6.09 ± 0.40                              Scs 38 1× adapted to porcine PMNLs                                      Scs 38                                                                             4.6 × 10.sup.2                                                                   8     8  0    3/8    5.22 ± 0.52                             Scs 38                                                                             4.6 × 10.sup.2                                                                   9    14  0    3/9   5.23 ± 0.18                              Scs 38 5× adapted to porcine PMNLs                                      Scs 38                                                                             4.7-6.4 × 10.sup.1                                                               16    8  0     1/16 0.40                                        Scs 38                                                                             4.7-6.4 × 10.sup.1                                                               17   14  0     2/17 0.20 ± 0.19                              Scs 38 7× adapted to porcine PMNLs                                      Scs 38                                                                             4.3 × 10.sup.3                                                                   10    8  0     0/10 0.00                                        Scs 38                                                                             4.3 × 10.sup.3                                                                   10   14  0     0/10 0.00                                        Scs 38 13× isolated from porcine neutrophil lysosomes                   Scs 38                                                                             2.8 × 10.sup.1                                                                   8     8  0    0/8   0.00                                        Scs 38                                                                             2.8 × 10.sup.1                                                                   8    14  0    0/8   0.00                                        Scs 38                                                                             6.5 × 10.sup.2                                                                   8     8  0     1/10 3.00                                        Scs 38                                                                             2.5 × 10.sup.3                                                                   5    15  0    0/5   0.00                                        Scs 38                                                                             2.5 × 10.sup.4                                                                   5    15  0    0/5   0.00                                        Scs 38                                                                             2.5 × 10.sup.5                                                                   5    15  0    0/5   0.00                                        __________________________________________________________________________

                  TABLE 2                                                         ______________________________________                                        Lethal dose 50 (LD.sub.50) of Salmonellae and their PMNL adapted              derivatives. Adaptation of Salmonellae to PMNLs was described                 in Table 1. Groups of 5 mice each were inoculated with tenfold                increments of Salmonellae from 10.sup.1 to 10.sup.5 in the left footpad       in                                                                            a volume of 80 uL. Surviving mice were sacrificed 10 days after               inoculation, and LD.sub.50 calculated by the method of Reed and               Muench.                                                                                    LD.sub.50                                                        Salmonellae    wild      PMNL adapted                                         ______________________________________                                        S. choleraesuis str 38                                                                       1.0 × 10.sup.2.84                                                                 >2.1 × 10.sup.5                                S. dublin str 127                                                                            1.9 × 10.sup.2.33                                                                 >1.5 × 10.sup.5                                S. enteritidis str 129                                                                       1.9 × 10.sup.2.66                                                                 >2.1 × 10.sup.5                                ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Organ invasion of wild-type S. choleraesuis strain 38 (Scs 38) and            its PMNL-adapted derivative (Scs 38 7× PMNL) in pigs. Pigs              were fed respective cultures by gavage. All surviving pigs were               euthanized two weeks after infection and selected organs were                 cultured from tenfold dilutions of organ suspensions. The numbers             denote log.sub.10 of colony forming units. A single colony from the           1:10 (lowest) dilution was noted by "+".                                              Pigs                                                                          1   2     3     4   5   6   7    8   9    10                                    Scs 38 7× PMNL                                                Inocula   3.7 × 10.sup.9                                                                        Scs 38 3.2 × 10.sup.9                           ______________________________________                                        Tonsils   -     -     -   +   -   +   -    -   2.1  1.9                       Retro-    -     -     -   -   -   -   4.0  3.0 1.8  NA                        pharyngeal ln                                                                 Bronchial ln                                                                            -     -     -   -   -   -   5.7  1.1 3.6  4.5                       Mesenteric ln                                                                           -     -     -   -   -   -   3.7  1.1 4.3  4.1                       Ileo-caecal ln                                                                          -     +     -   -   -   +   3.9  1.8 3.9  3.6                       Lung      -     -     -   -   -   -   2.4  -   4.8  2.0                       Lung lesion                                                                             -     -     -   -   -   -   6.4  -   NA   2.2                       Liver     -     -     -   -   -   -   -    -   5.5  1.4                       Liver lesion                                                                            -     -     -   -   -   -   -    -   >6   NA                        Spleen    -     -     -   -   -   -   -    -   5.5  -                         Ileum     +     +     -   +   -   -   1.1  +   4.7  2.5                       Colon     +     -     -   +   -   +   2.9  -   4.2  1.4                                                             died     died died                      ______________________________________                                    

                                      TABLE 4                                     __________________________________________________________________________    Effects of wild type S. dublin and S. enteritidis and of their porcine        PMNL-adapted                                                                  derivates on mice. The PMNL selection process was the same as described       for                                                                           S. choleraesuis in Table 1.                                                                    Killed   No spleens                                                                          Log.sub.10                                    Inocula:                                                                             Dose No Mice                                                                            days pi                                                                           No Dead                                                                            infected                                                                            bacteria/spleen                               __________________________________________________________________________    S. dublin                                                                            8.9 × 10.sup.1                                                               10   9   1    10/10 7.17 ± 0.28                                S. dublin                                                                            1.2 × 10.sup.2                                                               10   9   0    3/0   5.60 ± 0.22                                5× PMN                                                                  S. enteritidis                                                                       9.6 × 10.sup.1                                                               10   9   2    10/10 7.22 ± 0.32                                S. enteritidis                                                                       1.2 × 10.sup.2                                                               10   9   0     4/10 5.01 ± 0.70                                5× PMN                                                                  __________________________________________________________________________

                                      TABLE 5                                     __________________________________________________________________________    Immunizing effect of S. choleraesuis strain 38 adapted to porcine PMNLs       (Scs 38 7× PMNL or 7×                                             PMNL). Three groups of 10 mice each were given footpad injections of Scs      38 7× PMNL in tenfold                                                   incremental doses (left column). Three weeks later, all 3 groups, and an      additional challenge control                                                  group were given footpad injections of 1.6 × 10.sup.3 Scs 38. Scs       38 7× PMNL carried a xylose.sup.+                                       marker, and thus enabled the separate identification of spleens and           colonies infected with the immunizing                                         strain (right columns).                                                       Scs injected  No Days                                                                             No No spleens infected                                                                     Spleen CFU counts                            Scs 38 7× PMNL                                                                   Scs 38                                                                             mice                                                                             pi dead                                                                             Scs 38                                                                            7× PMNL                                                                       Log.sub.10 Scs 38                                                                    log.sub.10 7× PMNL              __________________________________________________________________________    2.0 × 10.sup.1                                                                   1.6 × 10.sup.3                                                               10 8  2  5/8 2/8   2.90 ± 0.83                                                                       0.50 ±  0.12                       2.0 × 10.sup.2                                                                   1.6 × 10.sup.3                                                               10 8  0  3/10                                                                              5/10  1.40 ± 0.51                                                                       0.62 ± 0.19                        2.0 × 10.sup.3                                                                   1.6 × 10.sup.3                                                               10 8  0  2/10                                                                              1/10  0.82 ± 0.55                                                                       0.33  NA.sup.                         NA       1.6 × 10.sup.3                                                               10 8  4  10/10                                                                             NA    6.09 ± 0.52                                                                       NA                                    __________________________________________________________________________

EXAMPLE 2 Vaccination of Swine with an Attenuated Strain of Salmonellacholeraesuis

Materials and Methods

Attenuated Scs 38 strains prepared according to the procedure in theabove-described example were used in the following experiments. Anattenuated Scs 38 strain subjected to PMNL exposure five times wasdesignated Scs 54.

Mouse experiments. Female Swiss Webster mice approximately 6 weeks oldwere inoculated in the left footpad with either the avirulent orvirulent Scs 38 strains at varying dosages. The mice were killed bycervical dislocation 8 or 12 days after inoculation with Salmonella.Their spleens were excised aseptically, homogenized and diluted byserial tenfold dilutions, and each dilution plated on MacConkey agar.

Pig vaccine safety experiment. Ten pigs weighing approximately 20 kgwere purchased from an Iowa State University herd with no history ofclinical salmonellosis. Rectal samples from these pigs were culturedtwice for Salmonellae, and no Salmonellae were recovered. Baselinetemperatures were also determined on two occasions prior to the start ofthe experiment. Five of the pigs were randomly assigned to the vaccinesafety test group, and were given 3.7×10⁹ colony forming units (CFUs) ofScs 54 by gavage. The other five pigs were assigned to the challengecontrol group and were given 3.2×10⁹ Scs 38 CFUs by gavage. of thesurviving pigs were killed 14 days after Salmonella treatment. Tenorgans or lymphoid tissues were cultured quantitatively for Salmonella.

Vaccine Efficacy Experiment 1. This vaccine efficacy experimentconsisted of a double blind study involving nursery pigs (barrows andgilts) from an Iowa farm with a history of severe current swineparatyphoid with multiple bacteriologic diagnoses at the time of thestart of this experiment. All of the pigs were inoculated bynasopharyngeal gavage. One randomly selected group of 23 pigs was given2.2×10⁸ colony forming units (CFU) of Scs 54. A second group of 22 pigswas given 2.2×10⁸ autoclaved Scs 54. A third group of 22 pigs was givena starch suspension (placebo) adjusted to the same optical density asthe Scs suspensions. The groups were identified by ear notching, and allpigs were co-mingled Bottles were labeled "A", "B", and "C" and theingredient in each bottle was revealed to the owner and attendingveterinarian at the conclusion of the experiment only. All of the pigswere examined daily, and were weighed on the day of the trial, and 18days later.

Vaccine Efficacy Experiment 2. Ten pigs were randomly selected from agroup of pigs vaccinated per os with 1.0×10⁹ CFUs of Scs 54 and wereco-mingled with 6 healthy unvaccinated control pigs 20 days aftervaccination. All 16 pigs were given 2.0×10⁹ CFU of a virulent Scholeraesuis field isolate (challenge) by gavage. For reasons unrelatedto salmonellosis, one vaccinated and one control pig were later excludedfrom the experiment. Rectal temperatures were taken twice beforechallenge exposure and on days 2, 4, 5, 6, 7, 11, and 13 afterchallenge. All pigs were weighed two days before challenge exposure and7 and 14 days after challenge exposure. All challenge control pigs, and5 randomly selected vaccinated and challenged pigs were killed andnecropsied 14 days after challenge exposure. Bacteriologic cultures weredone on 6 organs of the challenge control pigs, and on 10 organs of thevaccinated and challenged pigs.

Differences between group means were evaluated by appropriate student ttests, and differences between count data were evaluated by chi-squaretests.

Field trials. Scs strain 54 was given to several thousand pigs indrinking water on 8 Iowa Farms with current acute and endemicsalmonellosis with multiple bacteriologically confirmed diagnoses. On 2farms, vaccinated during the summer and fall of 1990, the averageconcentration of Scs 54 was approximately 10⁷ CFU/dose, whereas fromDec. 7, 1990 the dose was increased to 10⁹ CFU/dose. On the average, thepigs weighed about 5-6 kgs., thus, the preferred dose to be administeredis about 10⁷ -10⁹ bacteria/Kg of body weight.

Results

Mouse virulence experiments. Increased numbers of virulent Scs 38 causedincreased mortality and spleen infections in mice; however, spleen CFUsremained relatively constant at 10⁶ and were dose independent. See Table6. By contrast, Scs 38 exposed once to neutrophils (Scs 38 PMNa-1×)caused no mortality and reduced spleen colonization, as well as having atenfold reduction of splenic CFUs. When Scs 38 was exposed five times toneutrophils (Scs38 PMNa-5×), it had almost totally lost virulence andinvasiveness. Similar loss of lethality, virulence and invasiveness wasachieved by adapting Scs 38 to lysosomal extracts (PMNlys-13×) ofporcine neutrophils. See Table 6.

Mouse immunization experiments. Three groups of 10 mice were eachinjected with 10-fold increments of Scs 54 (38PMNa-5×); 21 days laterthey, and a control group of 10 mice, were injected with 1.6×10³virulent Scs 38. See Table 7. Protection from challenge, judged byreduced mortality and spleen colonization was obtained in adose-dependent fashion, i.e., when mice were immunized with 2.0×10³ Scs54, none died after challenge and only 2 of 10 had spleens colonizedwith the challenge strain at very low level (0.82 log₁₀ CFUs). The countdata i.e., deaths and number of spleens colonized, were significantlydifferent between groups of mice immunized with 2.0×10² or higher Scs 54and challenge control groups (p<0.001).

Pig vaccine safety experiment. When the neutrophil adapted mouseavirulent Scs 54 was given to five pigs, a mild temperature riseoccurred on day 4 p.i. Five pigs were given a similar dose of thevirulent Scs 38 parent strain and had highly elevated temperatures fromp.i. days 2 through 8 and 3 of the 5 pigs died during this period. SeeFIG. 1. All of the pigs given the avirulent Scs 54 strain remained ingood health and were killed 14 days after treatment. Only a few coloniesof Scs 54 were isolated from the tonsils, ileocecal lymph nodes, ileumand colon of a few of these pigs, i.e., 7/50 organ suspensions (14%)yielded a few colonies from the undiluted organ suspensions. See Table8. By contrast, 3 of the 5 pigs infected with the virulent Scs 38 strainhad high bacterial counts in multiple organs and died, while the 2remaining controls were moderately infected. See Table 8. The number oforgans infected in the control group of pigs was 33/49 (67%).

Pig vaccine efficacy experiment 1. The pigs given 2.2×10⁸ Scs 54 gainedan average daily weight of at least 100 g in excess to pigs in either ofthe two control groups. There were no death losses among vaccinated pigsand none of the 23 vaccinated pigs required parenteral antibiotictreatment during the 18 day observation period. One pig died ofsalmonellosis in each of the 2 control groups of 22 pigs, and 3 pigsrequired parenteral treatment of gentamicin for acute septicemicsalmonellosis during the observation period. See Table 9.

Pig vaccine efficacy experiment 2. The vaccinated pigs challenged with2.0×10⁹ virulent S. choleresuis field strain had a single rectaltemperature rise to 40.4° C. 6 days after challenge. The temperaturerise subsided on the next day. The challenge control pigs had asustained temperature rise to 40.6° C. and higher from 5 to 13 daysafter vaccination. Three of these pigs died during the 14 dayobservation period. See FIG. 2. The average daily weight gains ofvaccinated and challenged pigs were 331 g, while the challenge controlpigs lost weight during the first week after challenge. Thecorresponding values for the second week were 344 g for the vaccinatedand challenged group and -54 g for the challenge control group. See FIG.3. Rectal temperature differences between the 2 groups from day 5 to 13and weight differences for the 2-week observation were significantlydifferent (p<0.01). The challenge virulent S. choeraesuis field strainwas isolated from the lungs, spleens, kidneys and mesenteric lymph nodesof all challenge control pigs. See Table 10. All spleen, kidney, ileum,ileocecal lymph node, colon and colonic lymph node cultures werenegative for Salmonella in the vaccinated and challenged group of pigs.Overall, 28/30 organs (93%) from the challenge control group, and 7/50organs (14%) from the vaccinated and challenged group yieldedSalmonella. See Table 10. This organ count difference was significant atp<0.01.

Field trials. On the farms with endemic salmonellosis when Scs 54 wasgiven in the drinking water at a concentration of 10⁷ CFU/dose,salmonellosis occurred several weeks after vaccination. When theconcentration of Scs 54 was raised to 10⁹ CFU/dose, no further outbreaksof salmonellosis have occurred. In one instance, 150 pigs were given thevaccine in the drinking water at 10⁹ CFUs/dose in the face of anoutbreak. Six pigs have died of salmonellosis on the days immediatelypreceding and 2 days following vaccination. The diagnosis was confirmedby Salmonella cultures from the lungs, livers, and lymph nodes of 3 deadpigs. Fifteen pigs had rectal temperatures between 40° and 42° C. twoand three days after vaccination. There were no further losses orclinical signs of salmonellosis in this group of pigs from the 4th dayafter vaccination in the drinking water. There were no clinical,pathological or microbiological diagnoses of salmonellosis made on the 8farms for the last 6 months since vaccination in the drinking water at aconcentration of 10⁹ CFU/dose was implemented. These observationssuggest that Scs 54 is effective and stable under field conditions, anddoes not revert to virulence.

Discussion

Observations with Scs 54 indicate that oral immunization of swine withthis strain is very effective, and that the loss of the 50 kb plasmiddid not prevent excellent immunogenicity in mice when vaccinated andchallenged parenterally and in pigs vaccinated and challenged per os.While the mechanism(s) of immunogenicity of Scs 54 are unknown at thepresent time, the strain appears to be preferentially adapted to lifewithin neutrophils. Salmonella choleraesuis strain 54 has effectivelystopped salmonellosis in field trials involving 8 large swine herds andseveral thousand pigs with no reversion to virulence when the vaccinewas given per os at a dose of 10⁹ CFU.

                                      TABLE 6                                     __________________________________________________________________________    Virulence of S. choleraesuis 38 and porcine neutrophil adapted                derivatives in Swiss                                                          Webster mice. Data were pooled from 4 experiments.                                                      % spleens                                                                           Log.sub.10 Scs 38/                            Inocula                                                                              Dose     No Mice                                                                            % Dead                                                                             infected                                                                            spleen                                        __________________________________________________________________________    Scs 38 1.6-5.0 × 10.sup.1                                                               27   11   85    6.96 ± 1.12                                       1.6-5.0 × 10.sup.2                                                               35   20   91    6.10 ± 0.92                                       1.6 × 10.sup.2                                                                   10   40   100   6.09 ± 0.40                                Scs 38 4.610.sup.2                                                                            17   0    35    5.22 ± 0.52                                PMNa-1×                                                                 Scs 38 4.7-6.4 × 10.sup.1                                                               33   0    9     0.30 ± 0.19                                PMNa-5×                                                                        4.3 × 10.sup.3                                                                   20   0    0     0.00                                          Scs 38 2.8 × 10.sup.1                                                                   16   0    0     0.00                                          PMNlys-13×                                                                     6.5 × 10.sup.2                                                                    8   0    12    3.00                                                 2.5 ×  10.sup.3                                                                   5   0    0     0.00                                                 2.5 × 10.sup.4                                                                    5   0    0     0.00                                                 2.5 × 10.sup.5                                                                    5   0    0     0.00                                          __________________________________________________________________________

                  TABLE 7                                                         ______________________________________                                        Immunizing effect of porcine neutrophil adapted S. choleraesuis               strain 54                                                                     Salmonella                             Spleen                                 injected*   No     Days   No   No spleens                                                                            CFU Log.sub.10                         Scs 54 Scs 38   mice   pi   dead infected                                                                              Scs 38                               ______________________________________                                        2.0 × 10.sup.1                                                                 1.6 × 10.sup.3                                                                   10     8    2    5/8     2.90 ± 0.83                       2.0 × 10.sup.2                                                                 1.6 × 10.sup.3                                                                   10     8    0    3/10    1.04 ± 0.51                       2.0 × 10.sup.3                                                                 1.6 × 10.sup.3                                                                   10     8    0    2/10    0.82 ± 0.55                       NA     1.6 × 10.sup.3                                                                   10     8    4    10/10   6.09 ± 0.52                       ______________________________________                                         *Swiss Webster mice were first injected with Scs 54, and 21 days later        with the virulent challenge strain Scs 38. All live mice were killed on       day 8 p.i.                                                               

                  TABLE 8                                                         ______________________________________                                        Quantitative isolation of PMNL-adapted S choleraesuis (Scs 54)                and of virulent Scs 38 from organs of pigs two weeks after                    infection by gavage.                                                          Pigs                                                                          1         2     3     4    5   6   7    8   9     10                          Organs  Scs 54 3.7 × 10.sup.9                                                                  Scs 38 3.2 × 10.sup.9                            ______________________________________                                        Tonsils -     -     -    +*  -   +   -    -    2.1  1.9                       Retro-  -     -     -   -    -   -   4.0  3.0 1.8   ND                        pharyngeal                                                                    ln                                                                            Bronchial                                                                             -     -     -   -    -   -   5.7  1.1 3.6   4.5                       ln                                                                            Mesenteric                                                                            -     -     -   -    -   -   3.7  1.1 4.3   4.1                       ln                                                                            Ileo-caecal                                                                           -     +     -   -    -   +   3.9  1.8 3.9   3.6                       ln                                                                            Lung    -     -     -   -    -   -   6.4  -   4.8   2.1                       Liver   -     -     -   -    -   -   -    -   5.5   1.4                       Spleen  -     -     -   -    -   -   -    -   5.5   -                         Ileum   +     +     -   +    -   -   1.1  +   4.7   2.5                       Colon   +     -     -   +    -   +   2.9  -   4.2   1.4                                                            died     died  died                      ______________________________________                                         *A few Salmonella colonies from the undiluted organ suspension.                 Log.sub.10 of CFUs/g tissue from tenfold dilutions.                    

                  TABLE 9                                                         ______________________________________                                        Clinical Observations of pigs given 2.2 × 10.sup.8                      Salmonella choleraesuis strain Scs 54 per os, and 2 control groups                          Daily weight       Gentamicin*                                                gain               treatment for                                Experimental Group                                                                          (g)        Deaths  Salmonellosis                                ______________________________________                                        Vaccinates (Scs 54)                                                                         593        0       0                                            N = 23                                                                        Controls (Autoclaved                                                                        488        3       11                                           Scs) N = 22                                                                   Controls (starch)                                                                           453        2       9                                            N = 22                                                                        ______________________________________                                         *Pigs required 3day intramuscular gentamicin treatment for salmonella         septicemia.                                                              

                                      TABLE 10                                    __________________________________________________________________________    Organ cultures from pigs vaccinated with Scs 54 and challenged with 2.0       × 10.sup.8 virulent                                                     S choleraesuis, and from challenge control pigs                                          Pig Groups                                                                    Vaccinates     Challenge Controls                                  Pig Nos:   402                                                                              404                                                                              407                                                                              410                                                                              412                                                                              414                                                                              415*                                                                             417*                                                                             418                                                                              419*                                    __________________________________________________________________________    Organs                                                                        Lung       Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              Pos                                                                              Pos                                                                              Pos                                                                              Pos                                                                              Pos                                                                              Pos                                     Liver      Pos                                                                              Neg                                                                              Neg                                                                              Neg                                                                              Pos                                                                              Neg                                                                              Pos                                                                              Pos                                                                              Neg                                                                              Pos                                     Spleen     Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              Pos                                                                              Pos                                                                              Pos                                                                              Pos                                                                              Pos                                     Kidney     Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              Pos                                                                              Pos                                                                              Pos                                                                              Pos                                                                              Pos                                     Ileocec ln Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              ND ND ND ND ND                                      Jejunal ln Pos                                                                              Pos                                                                              Neg                                                                              Neg                                                                              Pos                                                                              Pos                                                                              Pos                                                                              Pos                                                                              Pos                                                                              Pos                                     Colonic ln Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              ND ND ND ND ND                                      Ileocec    Pos                                                                              Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              Pos                                                                              Pos                                                                              Pos                                                                              Pos                                                                              Pos                                     valve                                                                         Ileum      Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              ND ND ND ND ND                                      Colon      Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              Neg                                                                              ND ND ND ND ND                                      Ratio: organs                                                                            7/50 (14%)     28/30 (93%)                                         infected/total sampled                                                        __________________________________________________________________________     *Pigs died of salmonellosis. ND = not done. ln = lymph node              

EXAMPLE 3 Characterization of an Salmonella Choleraesuis IsolateFollowing Repeated Neutrophil Exposure

The purpose of this investigation was to determine the changesassociated with Salmonella virulence following exposure to porcineneutrophils. Strain 38 was used in this investigation because of itsvirulence, and ability to ferment glycerol (+) which was used as amarker for recovery. This has been evaluated by measuring virulence in amouse model, resistance to phagocyte killing., plasmid analysis andinvasion.

Materials and Methods

Bacterial strains and cultures. Bacterial isolates were grown onMacConkey agar (Difco Laboratories, Detroit, Mich.) and pure culturesisolated. Cultures were stored on trypticase soy agar (TSA) (Difco) softagar slants at room temperature, and in trypticase soy broth (TSB)(Difco) which contained 15% glycerol at -70° C. Routine growth ofbacterial isolates was performed on TSA at 37° C. for 12 h. Bacterialstrains E. coli MC1040-2 and LE392 were propagated in Luria broth or insome cases in super broth (32 g tryptone, 20 g yeast extract, 5 g sodiumchloride per liter). Descriptions of genotypes are presented in Table11.

Cell culture. Vero cells were obtained from the National VeterinaryServices Laboratory U.S.D.A. The Vero cells were grown in Dulbecco'sminimum essential media (DMEM) (Sigma Chemical Co., St. Louis, Mo.) with10% fetal calf serum (Hyclone Laboratories Inc., Logan, Utah), at 37° C.with 5% CO₂ until confluent.

Neutrophil isolation. Porcine neutrophils were obtained from freshlycollected, anticoagulated blood from normal pigs. The neutrophils wereisolated using a Percoll gradient procedure as previously described. SeeRoof et al., Vet. Immuno. Immunopathol, 23:365-376 (1988) which isincorporated in its entirety by reference.

Neutrophil passage of S. Choleraesuis. An overnight culture of S.choleraesuis 38 grown on MacConkey agar was exposed to isolated porcineneutrophils in a bactericidal assay as previously described. See Roof etal., Vet. Immunol. Immunopathol., 23:365-376 (1989). Opsonized S.choleraesuis were incubated in M199 (Sigma) with neutrophils for 30 minto allow ingestion. Gentamicin (100 ug/ml) and kanamycin (100 ug/ml)were added for one hour to kill all extracellular bacteria. Theneutrophils were allowed to settle and the supernatant was sampled toinsure killing of extracellular Salmonella. A separate sample of S.choleraesuis without neutrophils was also exposed to antibiotics tomeasure killing. The neutrophils were washed three times with phosphatebuffered saline (PBS; 0.15M NaCl in 0.015M phosphate buffer, pH 7.4),and resuspended to their initial volume. A 100 ul aliquot was removedand lysed with distilled water to release intracellular bacteria. Thissample was then diluted and plated on MacConkey media and grownovernight at 37° C. Individual colonies were selected and resuspended to2×10⁸ colony forming units (CFU)/ml. This procedure was repeated fivetimes to enrich for a population of neutrophil-resistant S.choleraesuis. After the fifth passage, a single glycerol (+) clone wasselected for observation and designated S. choleraesuis 38 PMNa-5×.

Mouse infection with S. choleraesuis. Bacteria were suspended to 2×10⁸CFU/ml in PBS and tenfold dilutions from 10¹ to 10⁵ CFU/ml were injectedintraperitoneal (i.p.) into Balb/c mice as described. See Dulbecco,Microbiology, pg. 791 (1990), which is incorporated herein in itsentirety by reference.

S. Kholeraesuis susceptibility to neutrophil killing. The susceptibilityof S. choleraesuis 38 and 38 PMNa-5× to neutrophil killing was assessedby two methods: 1) A modification of a colorimetric assay that measuredthe ability of live bacteria to reduce3-[4,5-dimethylthiazol-2-yl]-2,5-diphenytetrazolium bromide (MTT) topurple formazan was employed. See Stevens et al., Vet. Immunol.Immunopthol., 27:accepted (1991), which is incorporated herein in itsentirety by reference. Opsonized S. choleraesuis (2×10⁸ /ml) in RPMl 11640 (Sigma) with 10% FCS (Hyclone) was incubated for 1 h with 1×10⁷neutrophils/ml. Following incubation, 0.2% Saponin was added to lyse theneutrophils. The remaining live S. choleraesuis were allowed to reduceMTT and then compared to a standard curve to determine the numbers ofviable S. choleraesuis. 2) A hydrogen peroxide sensitivity test wasperformed comparing the viability of S. choleraesuis 38 and PMNa-5×after exposure to 60 mM hydrogen peroxide for 1 hour. See Christman etal., Cell, 41:753-762 (1985), which is incorporated herein in itsentirety by reference.

Determination of Salmonella invasion of Vero Cells. The invasion assaywas performed in eight well plates as previously described. See Roof etal., Vet. Microbiol., accepted 1991, which is incorporated herein in itsentirety by reference. Briefly, freshly prepared Salmonellae (500 ul)were added to each well in a ratio of 1000:1 bacteria (2×10⁸) to Verocells (2×10⁵). After incubation for 2 hours, the wells were washed 3times with DMEM containing 100 ug/ml gentamicin to kill allextracellular bacteria. Controls containing bacteria only were performedat the same time to measure the killing of extracellular bacteria. Thewells were incubated for an additional 2 hours with DMEM containinggentamicin. The supernatants were examined for sterility by plating ontobrain heart infusion agar (BHl) (Difco). The wells were washed anadditional 3 times with DMEM to remove gentamicin, and the cellmonolayers were lysed using 0.1% sodium dodecyl sulfate, releasing allintracellular bacteria. Samples from each well were then diluted in PBSand plated in quadruplicate to determine the CFU. Results were expressedas the mean and standard error of the mean of five experiments.

Plasmid analysis. Plasmid DNA was isolated by an alkaline lysis method(1) from overnight cultures grown in super broth and purified by anethidium bromide-cesium chloride gradient as previously described. SeeSawbrook et al., Molecular Cloning: A Laboratory Manual, (1989), whichis incorporated herein in its entirety by reference. Plasmid analysiswas performed using a 0.5% agarose gel in Tris-borate buffer, run for 4hours at 70 mV and stained with ethidium bromide. Southern blots wereperformed using nylon membranes (Schleicher & Schuell Inc., Keene, N.H.)and standard methodology. The 50 kilobase plasmid of S. choleraesuis wasexcised from the gel, isolated using Geneclean II (Bio101 Inc., LaJolla, Calif.), and labeled with ³² P using oligonucleotide labeling(Amersham Corp., Arlington Heights, Ill.).

The introduction of the 50 kb plasmid into the cured strain 38 PMNa-5×was accomplished by labeling the plasmid from strain 38 with mini-Mu,transforming into the recipient and selecting for kanamycin resistance.See Groisman et al., J. Bacteriol., 168:357-364 (1986), which isincorporated herein in its entirety by reference. Lysates of mini-Muphage were prepared by thermoinduction of MC1040-2 which containedmini-Mu as a plasmid replicon and also Mucts62 temperature-sensitivehelper phage in the chromosome. Infections were performed by mixingdilutions of phage with mid-log phase culture of strain 38. Following a2 hour incubation, cells were plated on kanamycin (10 ug/ml) containingagar. Resistant colonies were collected in bulk and grown overnight insuper broth at 37° C. and the plasmid DNA prepared as described above.The plasmid was isolated by electroelution from agarose slices using anElutrap (Schleicher & Schuell) run at 200 mV for 18 hours. Strain 38PMNa-5× was electroporated with this plasmid preparation, using a BTX100 power supply (San Diego, Calif.) at 725 mV for 5 msec and anelectrode gap of 0.5 mm. The bacteria were then plated on kanamycinagar. Plasmid DNA was isolated from kanamycin resistant colonies, andanalyzed by DNA-DNA hybridization as described above.

Complement sensitivity. Normal porcine and guinea pig sera were obtainedby venipuncture, aliquoted and stored at -20° C. These sera did notagglutinate Salmonella O and H antigens. The complement assay wasconducted as described elsewhere. See Moll et al., FEMS Microbiol.Lett., 6:273-276 (1979), which is incorporated herein in its entirety byreference. Briefly, overnight cultures of Salmonella were diluted 1:100in TSB and incubated at 37° C. until they reached an O.D. of 0.20 at 540nm (2×10⁸ cfu/ml.). The bacteria were then centrifuged at 5000×g for 10min and resuspended in PBS. Bacteria (500 ul) were then added to 2 ml ofPBS containing serum at concentrations ranging from 10-50%. Thebacterial suspensions were then incubated at 37° C. and samples taken at0 and 90 min, diluted in PBS, and plated on MacConkey agar for viablecounts.

Carbohydrate and enzymatic activity. S. choleraesuis strain 38 and 38PMNA-5× were examined by API-CHE for fermentation of 49 substrates, andfor 19 enzyme activities by API-ZYME (API Analytab Prod., Plainview,N.Y.).

Statistical analysis. Data were analyzed by the Student's t test aspreviously described. See Zar, J. H., Biostatistical Analysis, (1984),which is incorporated herein in its entirety by reference. Allexperiments were carried out in at least triplicate with reproducibleresults.

Results

S. choleraesuis virulence for mice. The parent strain 38 was virulent inSwiss-Webster and Balb/c mice by footpad and i.p. injection with an LD₅₀of 10².84 in Swiss-Webster mice. The PMN-adapted derivative, 38 PMNa-5×had a LD₅₀ of greater than 10⁵ in mice. The pathogenicity of Salmonellafor mice was evaluated based on death, spleen infection and number ofrecovered bacteria. The parent strain 38 caused 100% death, 100% spleeninfection, and Log₁₀ 8.4 bacteria per spleen, but strain 38 PMNa-5×failed to infect spleens or kill mice. See Table 12. Strain 38 PMNa-5×had been cured of a 50 kb plasmid, and following re-insertion of akanamycin-marked plasmid, virulence was partially restored. Death ratesof the resulting strains (38-K28, 38-K65, and 38-K71) ranged from 16-66%with 100% infection of spleens and bacterial recovery intermediatebetween strain 38 and 38 PMNa-5×. See Table 12.

Viability of S. choleraesuis 38 and 38 PMNa-5× following PMN andhydrogen peroxide exposure. The overall viability of these two isolatesfollowing neutrophil exposure and ingestion was determinedcolorimetrically by measuring the reduction of MTT to formazan. Theparent isolate 38 remained 32% viable after 1 hour of neutrophilexposure compared to 56% viability for the 38 PMNa-5× isolate. See Table13. Strains 38-K28, K65, and K71 were intermediate in resistance toneutrophil killing. See Table 13. Viability was also examined afterexposure to hydrogen peroxide. S. choleraesuis was exposed to 60 mMhydrogen peroxide for 1 hour and then viable numbers of bacteriadetermined by plating and counting CFU. The parent isolate 38 remained50% viable after one hour compared to 65% viable for the PMN adapted 38PMNa-5×. See Table 13. Strains 38-K28, K65, and K71 were intermediate insusceptibility to hydrogen peroxide killing. See Table 13.

DNA analysis. Plasmid DNA was isolated from S. choleraesuis before andafter neutrophil passage. Following the PMN-adaptation process, theisolate was cured of its large virulence plasmid. This was confirmed byDNA-DNA hybridization using a ³² P-labeled virulence plasmid from theparent strain as a specific probe. To determine the role of thevirulence plasmid in the phenotypic changes noted followingneutrophil-adaptation, strain 38 PMNa-5× was re-introduced with a 50 kbplasmid from the parent strain. This was accomplished by marking theparent plasmid with a Mu derivative coding for kanamycin resistance. SeeGroisman et al., J. Bacteriol., 168:357-364 (1986). The marked plasmidwas introduced by electroporation and presence of the plasmid wasconfirmed by DNA-DNA hybridization. Thirteen kanamycin resistantderivatives (K-strains) of strain 38 PMNa-5× were isolated by mini-Mutransfection and placed into mice to study their virulence potential. Ofthese, three were recovered from mice after 14 days, 38-K28, 38-K65, and38-K71. These three strains displayed intermediate virulence between theparent strain and the neutrophil adapted strain 38 PMNa-5× usingmortality, spleen invasion, and the number of recovered bacteria as thecriteria for virulence. See Table 12. They were recovered from 100% ofthe mouse spleens in intermediate numbers, and had death rates rangingfrom 16-66%. See Table 12. In these strains, the plasmid was stablymaintained during the in vivo passage.

Invasion of Vero cell monolayers by S. choleraesuis 38 and 38 PMNa-5×.To determine the invasiveness of these isolates, confluent Vero cellmonolayers were incubated with live S. choleraesuis as previouslydescribed. S. choleraesuis 38 were recovered at a rate significantlyhigher than 38 PMNa-5× (3.8 vs 1.1). See Table 13. Strains 38-K28,38-K65, and 38-K71 had their invasive capabilities restored by there-introduction of the 50 kb plasmid. See Table 13.

Complement sensitivity. Both isolates were resistant to 50% fresh normalporcine and guinea pig sera. No difference could be noted by opticaldensity of growth or recovery of CFU/ml from the serum sample. Theparent strain was 95±3.0% viable and the attenuated 38 PMNa-5× was96±2.2% viable after 90 min of serum exposure.

Carbohydrate and enzymatic activity. Carbohydrate utilization wasexamined by the API-CHE system for enteric bacteria. Forty-ninecarbohydrates were examined and both isolates fermented the same fifteenof forty-nine samples. One of the fifteen carbohydrates fermented byboth 38 and 38 PMNa-5× was glycerol. Glycerol is not typically fermentedby S. choleraesuis and allowed differentiation from all other laboratoryand field isolates examined. Investigations defining typical S.choleraesuis fermentation patterns report no (0% glycerol fermentation.See Ewing, W. H., Enterobacteriaceae (1986), which is incorporatedherein in its entirety by reference. The API-CHE (API Analytab Prod.,Plainview, N.Y.) also indicated no (negative (-)) glycerol fermentationby S. choleraesuis in their standard identification profile. Thisallowed glycerol fermentation to be used as an identification marker.Strain 38 PMNa-5× was serologically and biochemically identical to theparental strain 38, and was distinctly different from all other isolatesof S. choleraesuis examined. Examination of glycerol negative isolatesfound conversion to glycerol positive to be less than one in 10⁸ CFU/ml.Enzymatic activity was examined using the API-ZYME system with bothisolates positive for the same 5 of 19 substrates (alkaline phosphatase,esterase-lipase, leucine amino-peptidase, acid phosphatases, andphosphohydrolase). Both strain 38 and strain 38 PMNa-5× had API-Eidentification No. 4504510.

Discussion

The pathogenesis of S. choleraesuis depends on its ability to invade theintestinal epithelium, survive the host immune response, and thendisseminate in the host. The ability of Salmonella to resist phagocytekilling has been well documented, but only recently has the role ofenvironmental induction of bacterial determinants been investigated.

The hypothesis of this research was that during infection, S.choleraesuis are repeatedly exposed to ingestion and killing by hostphagocytes. During this process, intracellular environmental stimulifrom the phagocytes might induce genetic or phenotypic changes in theviable S. choleraesuis. Our goal was to mimic these conditions in vitroand identify changes associated with porcine neutrophil association.

Following neutrophil exposure, isolates had increased resistance tokilling by neutrophils as well as resistance to hydrogen peroxide. Thismay have been due to selection for isolates which constituitivelyexpress resistance proteins, similar to OxyR and phoP as described withS. typhimurium. See Storz et al., Science, 248:189-194 (1990), Fields etal., Science, 243:1059-1062 (1989) and Groisman et al., Proc. Natl.Acad. USA, 86:7077-7081 (1989), all of which are incorporated herein intheir entirety by reference.

The loss of virulence in these isolates for mice was unexpected. Onepossible explanation may be the loss of the S. choleraesuis 50 kbplasmid previously shown to be involved in the invasion process andvirulence. See Gulig et al., Infect. Immun., 55:2891-2901 (1987), whichis incorporated herein in its entirety by reference. Our data supportthis conclusion since the re-introduction and stable maintenance of theplasmid resulted in partial restoration of virulence. See Table 12.Because full virulence was not restored, other genetic changes may havealso occurred in the neutrophil-adapted strain. This may correlate withinduction of stress- or heat-shock proteins beneficial for survivalinside neutrophils, but this study did not address this possibility.Sixteen of 19 K-strains did not maintain the virulence plasmid in-vitroand subsequently were not recovered from mice. This suggests that agenetic lesion(s) had occurred in the neutrophil-adapted strain in agene(s) involved in plasmid replication. The stable K-strains would thuscontain revertants or suppressor mutations that support maintenance ofthe virulence plasmid. Since, total virulence was not restored, otherundefined changes also may have occurred in the neutrophil adaptedstrain.

In conclusion, the following changes were noted in S. choleraesuisfollowing repeated PMN exposure: 1) There was an overall increasedresistance to neutrophil killing and killing by hydrogen peroxide, 2)the neutrophil adapted S. choleraesuis lost the large virulence plasmid,3) the virulence of the neutrophil adapted isolate (38 PMNa-5×) for micewas decreased, 4) S. choleraesuis 38 PMNa-5× was non-invasive in a Verocell assay, and 5) partial restoration of S. choleraesuis 38 PMNa-5×virulence and invasiveness was obtained by re-insertion of the 50 Kbplasmid.

                  TABLE 11                                                        ______________________________________                                        Bacterial strains                                                             Strain   Description       Source or reference                                ______________________________________                                        38       S. choleraesuis wild type                                                                       1, 7, 20, 28, 29                                            (glycerol +)                                                         38PMNa-5×                                                                        S. choleraesuis neutrophil                                                                      this work                                                   adapted 5 passages                                                            (glycerol +)                                                         38-K28   S. choleraesuis 38PMNa-5×                                                                 this work                                                   mini-Mu (Km.sup.r) marked                                                     plasmid                                                              38-K65   S. choleraesuis 38PMNa-5×                                                                 this work                                                   mini-Mu (Km.sup.r) marked                                                     plasmid                                                              38-K71   S. choleraesuis 38PMNa-5×                                                                 this work                                                   mini-Mu (Km.sup.r) marked                                                     plasmid                                                              X.sup.3246                                                                              S. choleraesuis wild type                                                                      Dr. Roy Curtis                                     MC1040-2 E. coli F.sup.-  araD169                                                                        21                                                          araB::Mu cts lacX74 galU                                                      galK rpsL, pEG5005                                                   LE392    E. coli F.sup.-  hsdR514 (r.sub.k -,                                                            F. C. Minion                                                m.sub.k) lacY1 supE44 galT22                                                  trpR55 metB1                                                         ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        Virulence of S. choleraesuis 38 and 38PMNa-5× in Balb/c mice            following intraperitoneal injection of 10.sup.2 bacteria for 14 days.                  # OF                    %      LOG.sub.10                                     BAC-     #       %      INFEC- CFU/                                  INOCULUM TERIA    MICE    DEATH  TION   SPLEEN                                ______________________________________                                        38       1.8 × 10.sup.2                                                                   6       100    100    8.4                                   38PMNa-5×                                                                        2.1 × 10.sup.2                                                                   12       0      0     0                                     38-K28   2.5 × 10.sup.2                                                                   6       66     100    4.4                                   38-K65   4.5 × 10.sup.2                                                                   6       16     100    4.1                                   38-K71   3.8 × 10.sup.2                                                                   6       50     100    4.2                                   ______________________________________                                    

                                      TABLE 13                                    __________________________________________________________________________    Comparison of S. choleraesuis and their resistance to neutrophil killing,     hydrogen                                                                      peroxide, and Vero cell invasion.                                                    MTT*    H.sub.2 O.sub.2.sup.#                                                                  VERO CELL INVASION.sup.                               Strain (%) Survival                                                                          (% Survival)                                                                           (Log.sub.10 cfu/ml)                                   __________________________________________________________________________    38      32.1 +/- 2.1.sup.§                                                              .sup. 50.5 +/- 0.8.sup.                                                                3.8 +/- 0.4                                           38PMNa-5×                                                                       56.2 +/- 2.9.sup.§                                                              .sup. 65.2 +/- 2/2.sup.                                                                .sup.  1.1 +/- 0.8.sup.                               38-K28 49.1 +/- 6.3                                                                          60.7 +/- 3.7                                                                           3.5 +/- 0.9                                           38-K65 48.7 +/- 5.5                                                                          58.6 +/- 4.1                                                                           3.5 +/- 1.1                                           38-K71 40.3 +/- 4.2                                                                          55.0 +/- 2.9                                                                           3.1 +/- 0.6                                           __________________________________________________________________________     (*) S. choleraesuis strains were evaluated for their resistance to            neutrophil killing after 1 h as measured by reduction of                      3[4,5═dimethylthiazol2-yl2,5-diphenyltetrazolium bromide (MTT).            (.sup.#) Isolate viability was examined after exposed to 60 mM hydrogen      peroxide for 1 h.                                                             (.sup. ) Vero cell invasion was determined by exposing S. choleraesuis        strains to confluent Vero cell monolayers for 2 h, and killing of             extracellular bacteria with gentamicin (100 ug/ml). Viable bacteria were      recovered and expressed as Log10 cfu/ml. Data (*, .sup.#, .sup. )             represents mean +/- sem of five experiments.                                  (.sup.§, .sup. )identical letters indicate significant differences (     < 0.01) between strains.                                                      (.sup.  )this isolate is significantly different (P < 0.01) than all othe     strains.                                                                 

I claim:
 1. A vaccine for inducing an immune response to Salmonellabacteria in a pig comprising Salmonella choleraesuis var. Kunzendorfstrain 38 PMNa, having ATCC accession number 55105, in apharmaceutically acceptable carrier, wherein said Salmonellacholeraesuis is in an amount effective to produce an immune response. 2.A method for inducing an immune response to Salmonella bacteria in a pigcomprising administering to said pig Salmonella choleraesuis var.Kunzendorf strain 38 PMNa, having ATCC accession number 55105, whereinsaid Salmonella choleraesuis is in an amount effective to produce animmune response.
 3. The method of claim 2 wherein said Salmonellacholeraesuis is administered in a pharmaceutically acceptable carrier.4. A method of making a vaccine for inducing an immune response toSalmonella bacteria in a pig comprising mixing Salmonella choleraesuisvar. Kunzendorf strain 38 PMNa, having ATCC accession number 55105, witha pharmaceutically acceptable carrier, wherein said Salmonellacholeraesuis is in an amount effective to produce an immune response.